These studies should enhance our understanding of the molecular basis for human alpha and beta chorionic gonadotropin (hCG) gene expression in normal and neoplastic tissues. The chromosomal location of human CG genes has been determined using somatic cell hybrids of known chromosomal content for assignment of hCG genes to specific chromosomes, together with Southern blotting of the hybrid cell DNA and labeled hCG cDNA probes. Natural alpha and beta hCG genes have been selected from human genomic libraries; their structures have been compared to those from normal placentae and trophoblastic and nontrophoblastic neoplasms to evaluate gene number and possible rearrangements, again by Southern blotting. Chromatin structure of hCG genes, as reflected by DNase sensitivity and methylation, have been elucidated in the various normal and neoplastic tissues. Specific sequences in the 5'-flanking region of alpha and beta hCG genes that may act as promoters of gene expression will be defined by testing specific deletion mutants of natural hCG genes in a cell-free transcription system. To study the nature of the mRNAs transcribed from alpha and beta CG genes in normal placentae, malignant trophoblastic cell lines, and malignant tumors secreting hCG or one of its subunits, we will determine the processing of nuclear precursors to mature cytoplasmic hCG mRNAs by the Northern blotting method. The effects of apparent stimulators of hCG production such as epidermal growth factor, sodium butyrate, and dibutyryl cAMP, are being investigated. The molecular level of the regulation will be elucidated: transcription initiation using isolated nuclei, mRNA processing (levels of nuclear and cytoplasmic mRNA), and translational efficiency as determined in the reticulocyte lysate translation system, as well as post-translational effects on alpha and hCG-beta protein. Lastly, human alpha and beta hCG chromosomal genes will be transferred into eukaryotic cells not expressing these genes to see whether expression will be obtained, including proper subunit processing and combination, when both genes are simultaneously introduced. Specific hCG gene deletion mutants will also be used to evaluate effects on subsequent expression. These studies should provide insights into the association of hCG production and neoplastic transformation, as well as increase our understanding of normal alpha and beta hCG gene expression. (C)